IDENTIFICATION OF THE HORSE EPIDERMAL GROWTH-FACTOR (EGF) CODING SEQUENCE AND ITS USE IN MONITORING EGF GENE-EXPRESSION IN THE ENDOMETRIUM OF THE PREGNANT MARE

The PCR technique and highly degenerate oligonucleotide primers were u sed to amplify a 282 bp fragment of the horse (Equus caballus) epiderm al growth factor (EGF) cDNA. The clone corresponded to 94 amino acids of the EGF precursor molecule. The deduced amino acid sequence of the 53 residue EGF mitogenic peptide within the precursor sequence showed 60-70% identity with five other published EGF sequences. The PCR cDNA fragment hybridized to a 4.9 kb transcript in horse kidney and endomet rial RNA which was of a similar size to the mature EGF transcript foun d in other mammalian species. The horse cDNA clone was used in Norther n blots to monitor EGF expression in the endometrium of pregnant mares up to day 83 of gestation (term = 330-340 days). The level of express ion increased from day 33 and showed a further dramatic increase betwe en days 35 and 45, which coincides with the onset of implantation and placentation in this species. Levels remained elevated up to day 83. T he horse DNA sequence was used to design sense and antisense oligonucl eotide probes (45-mers) for in situ hybridization studies. The antisen se probe showed specific hybridization to the glandular, but not lumen al, epithelial cells of the endometrium and there was no signal in fet al membranes. The in situ hybridization signal increased between days 35 and 45 to a similar degree to that observed in the Northern blot an alysis. This dramatic increase in EGF expression in the glandular epit helium of the mare's endometrium during pregnancy may provide a mitoge nic stimulus to the endometrium and/or trophoblast to facilitate place ntal differentiation and attachment. Alternatively, the precursor coul d be involved in the endometrial gland secretory process which is nece ssary to produce uterine milk for fetal sustenance. The PCR cloning me thods used in this study should be generally applicable to the cloning of EGF cDNAs from other species.